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Biochemistry 2/e - Garrett & Grisham
Chapter 26
Nitrogen Acquisition and Amino
Acid Metabolism
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
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Biochemistry 2/e - Garrett & Grisham
Outline
• 26.1 The Two Major Pathways of N
Acquisition
• 26.2 The Fate of Ammonium
• 26.3 Glutamine Synthetase
• 26.4 Amino Acid Biosynthesis
• 26.5 Metabolic Degradation of Amino
Acids
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Biochemistry 2/e - Garrett & Grisham
Major Pathways for N
Acquisition
• All biological compounds contain N in a
reduced form
• The principal inorganic forms of N are in an
oxidized state
• Thus, N acquisition must involve reduction of
the oxidized forms (N2 and NO3-) to NH4+
• Nearly all of this is in microorganisms and
green plants. Animals gain N through diet.
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Overview of N Acquisition
Nitrogen assimilation and nitrogen fixation
• Nitrate assimilation occurs in two steps:
2e- reduction of nitrate to nitrite and 6ereduction of nitrite to ammonium (page
854)
• Nitrate assimilation accounts for 99% of
N acquisition by the biosphere
• Nitrogen fixation involves reduction of
N2 in prokaryotes by nitrogenase
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Nitrate Assimilation
Electrons are transferred from NADH to nitrate
• Pathway involves -SH of enzyme, FAD,
cytochrome b and MoCo - all protein-bound
• Nitrate reductases are big - 210-270 kD
• See Figure 26.2 for MoCo structure
• MoCo required both for reductase activity
and for assembly of enzyme subunits to
active dimer
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Nitrite Reductase
Light drives reduction of ferredoxins and
electrons flow to 4Fe-4S and siroheme
and then to nitrite
• See Figure 26.2b for siroheme structure
• Nitrite is reduced to ammonium while
still bound to siroheme
• In higher plants, nitrite reductase is in
chloroplasts, but nitrate reductase is
cytosolic
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Enzymology of N fixation
•
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•
•
Only occurs in certain prokaryotes
Rhizobia fix nitrogen in symbiotic association
with leguminous plants
Rhizobia fix N for the plant and plant provides
Rhizobia with carbon substrates
All nitrogen fixing systems appear to be identical
They require nitrogenase, a reductant (reduced
ferredoxin), ATP, O-free conditions and
regulatory controls (ADP inhibits and NH4+
inhibits expression of nif genes
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Nitrogenase Complex
Two protein components: nitrogenase reductase
and nitrogenase
• Nitrogenase reductase is a 60 kD homodimer
with a single 4Fe-4S cluster
• Very oxygen-sensitive
• Binds MgATP
• 4ATP required per pair of electrons transferred
• Reduction of N2 to 2NH3 + H2 requires 4 pairs
of electrons, so 16 ATP are consumed per N2
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Why should nitrogenase need
ATP???
• N2 reduction to ammonia is
thermodynamically favorable
• However, the activation barrier for
breaking the N-N triple bond is
enormous
• 16 ATP provide the needed activation
energy
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Nitrogenase
A 220 kD heterotetramer
• Each molecule of enzyme contains 2
Mo, 32 Fe, 30 equivalents of acid-labile
sulfide (FeS clusters, etc)
• Four 4Fe-4S clusters plus two FeMoCo,
an iron-molybdenum cofactor
• Nitrogenase is slow - 12 e- pairs per
second, i.e., only three molecules of N2
per second
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The Fate of Ammonium
Three major reactions in all cells
• Carbamoyl-phosphate synthetase I
– two ATP required - one to activate bicarb,
one to phosphorylate carbamate
• Glutamate dehydrogenase
– reductive amination of alpha-ketoglutarate
to form glutamate
• Glutamine synthetase
– ATP-dependent amidation of gammacarboxyl of glutamate to glutamine
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Ammonium Assimilation
•
•
•
•
•
Two principal pathways
Principal route: GDH/GS in organisms rich in N
See Figure 26.11 - both steps assimilate N
Secondary route: GS/GOGAT in organisms
confronting N limitation
GOGAT is glutamate synthase or
glutamate:oxo-glutarate amino transferase
See Figures 26.12 and 26.13
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Glutamine Synthetase
A Case Study in Regulation
• GS in E. coli is regulated in three ways:
– Feedback inhibition
– Covalent modification (interconverts
between inactive and active forms)
– Regulation of gene expression and protein
synthesis control the amount of GS in cells
– But no such regulation occurs in eukaryotic
versions of GS
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Allosteric Regulation
of Glutamine Synthetase
• Nine different feedback inhibitors: Gly,
Ala, Ser, His, Trp, CTP, AMP,
carbamoyl-P and glucosamine-6-P
• Gly, Ala, Ser are indicator of amino acid
metabolism in cells
• Other six are end products of a
biochemical pathway
• This effectively controls glutamine’s
contributions to metabolism
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Covalent Modification
of Glutamine Synthetase
• Each subunit is adenylylated at Tyr-397
• Adenylylation inactivates GS
• Adenylyl transferase catalyzes both the
adenylylation and deadenylylation
• PII (regulatory protein) controls these
• AT:PIIA catalyzes adenylylation
• AT:PIID catalyzes deadenylylation
• -ketoglutarate and Gln also affect
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Gene Expression
regulates GS
• Gene GlnA is actively transcribed only if
transcriptional enhancer NRI is in its
phosphorylated form, NRI-P
• NRI is phosphorylated by NRII, a protein
kinase
• If NRII is complexed with PIIA it acts as a
phosphatase, not a kinase
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Amino Acid Biosynthesis
• Plants and microorganisms can make all 20
amino acids and all other needed N metabolites
• In these organisms, glutamate is the source of N,
via transamination (aminotransferase) reactions
• Mammals can make only 10 of the 20 aas
• The others are classed as "essential" amino acids
and must be obtained in the diet
• All amino acids are grouped into families
according to the intermediates that they are made
from - see Table 26.1
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The -Ketoglutarate Family
•
•
•
•
Glu, Gln, Pro, Arg, and sometimes Lys
Proline pathway is chemistry you have
seen before in various ways
Look at ornithine pathway to see the
similarity to the proline pathway
Note that CPS-I converts ornithine to
citrulline in the urea cycle (Figure 26.23)
Know the CPS-I mechanism - Figure
26.22
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The Urea Cycle
• N and C in the guanidino group of Arg
come from NH4+, HCO3- (carbamoyl-P),
and the -NH2 of Glu and Asp
• Breakdown of Arg in the urea cycle
releases two N and one C as urea
• Important N excretion mechanism in
livers of terrestrial vertebrates
• Urea cycle is linked to TCA by fumarate
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Lysine Biosynthesis
in some fungi and in Euglena
Lys derived from -ketoglutarate
Must add one C - it’s done as in TCA!
Transamination gives -aminoadipate
Adenylylation activates the -COOH for
reduction
• Reductive amination give saccharopine
• Oxidative cleavage yields lysine
•
•
•
•
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The Aspartate Family
Asp, Asn, Lys, Met, Thr, Ile
• Transamination of OAA gives Asp
• Amidation of Asp gives Asn
• Thr, Met, and Lys are made from Asp
(See Figure 26.27)
• -Aspartyl semialdehyde and
homoserine are branch points
• Note role of methionine in methylations
via S-adenosylmethionine (Fig. 26.28)
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The Pyruvate Family
Ala, Val, Leu
• Transamination of pyruvate gives Ala
• Val is derived from pyruvate
• Note that Ile synthesis from Thr mimics
Val synthesis from pyruvate (Fig. 26.29)
• Leu synthesis, like that of Ile and Val,
begins with an -keto acid
• Transaminations from Glu complete
each of these pathways (Figs. 26.29-30)
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3-Phosphoglycerate Family
Ser, Gly, Cys
• 3-Phosphoglycerate dehydrogenase
diverts 3-PG from glycolysis to aa paths
• Transamination by Glu gives 3-P-serine
• Phosphatase yields serine
• Serine hydroxymethylase (PLP)
transfers the -carbon of Ser to THF to
make glycine
• A PLP-dependent enzyme makes Cys
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Aromatic Amino Acids
Phe, Tyr, Trp, His
• Shikimate pathway yields Phe, Tyr, Trp
• Note the role of chorismate as a branch
point in this pathway (Figs. 26.36-7)
• Note the ‘channeling’ in tryptophan
synthase (Figure 26.39)
• His synthesis, like that of Trp, shares
metabolic intermediates with purine
biosynthetic pathway
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Degradation of Amino Acids
The 20 amino acids are degraded to
produce (mostly) TCA intermediates
• Know the classifications of amino acids
in Figure 26.41
• Know which are glucogenic and
ketogenic
• Know which are purely ketogenic
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